Therapeutic product and process



Patented Apr. 6, 1937:

' THERAPEUTIC PRODUCT AND PROCESS Hans Christian Hagedorn, Gentofte, Birger Norman Jensen, Copenhagen, and Ingrid Wodstrup-Nielsen, Gentofte,.Denmark, assignors to Nor-disk Insulinlaboratorium, Gentoite, Denmark, a corporation of Denmark No Drawing. Application February 14, 1936,

Serial No. 63,890.

8 Claims.

It is the object of our invention to control the rate at which Insulin becomes efiective in the body, and to obtain a more effective utilization of Insulin.

More specifically, the object is to make an eiiective Insulin product the solubility of which in the fluids of the body tissues is limited to a sugar-control approaches zero;

very low but controlled value, so that the In sulin will become effective at a-slow but predetermined rate over an extended period of time;

and thus to make the effect on administering such product to a patient not only more lasting but also more uniform between successive doses as compared with ordinary Insulin, and so to lessen the danger of having an Insulin effect. that is either inadequate or excessive at any time and thus to hold the blood sugar at all times more nearly like that of a normal person and incidentally to lessen thereby the total quantity of Insulin necessary per day for the eiiectlve control of diabetes. a

It is well known that the eifect of the usual administration of ordinary Insulin, (which is most commonly in the form of the hydrochloride) by subcutaneous or intramuscular injection from one to five times per day, is far from uniform between doses; for after each injection there tends to be for a time too great an Insulin effect in the blood stream and a corresponding tendency to hypoglycemia, while after a few hours the Insulin is dissipated and there is available too little Insulin effect and a consequent tendency to the diabetic condition of hyperglycemia.

The situation is the more serious in the more severe cases of diabetes, where the patients own cases, and to a less extent in milder cases, this has made necessary the administration of Insulin in smalldo'ses at inconveniently frequent intervals; for there is danger to the patient both from too large a dose and from too long a period between successive doses.

Many attempts have been made to counteract this situation, but with little success. Among the things which have been tried-are the injection of Insulin in the form of a suspension in oil; the application of Insulin, in variousjforms, by inunction for absorption through the skin; the slowing down of the absorption of Insulin by the use of epinephrine; and the injection of Insulin in the form of an emulsion with -yarious lipoids-,-,with or without'some emulsion-stabilizer. co-present. These are all open to serious objec- In such severe In Denmark April 8, 1935 tion from the standpoint of use in the human diabetic.

In addition, various compositions of Insulin, whether or not there was a chemical or physical combination or a mere mixing of the ingredients, have been tried, but'with very little success in meeting this situation prior to our present invention. I

Our invention may also be considered to approach the problem bythe use (at least in part) of what may be compounds of Insulin--but of a new type. According to that invention, we form a product (possibly at least in part a chemical compound) by mixing Insulin and. a protamine; and that product includes what may be called an Insulin-protamine composition, which at about the hydrogen ion concentration of the fluids of the body tissues is almost insoluble in such fluids, so that when such product is injected the Insulin istaken up by such fluids from the site of injection only very slowly and over a relatively long period of time. In this way, the peaks of Insulin-effect are lowered and the valleys of Insulin-efiect are raised; both the danger of hypoglycemia and the danger of hyperglycemia are greatly lessened; and the amount administered at a dose may with safety be increased and the time between successive injections may with safety be lengthened. By proper selection of the protamine, and of its concentration, the rate of absorption of the Insulin into the fluids of the body tissues may be controlled with considerable effectiveness.

We have found that protamines somehow combine with Insulin to form products, possibly chemical or physical compounds of some nature,

which when at or near the hydrogen ion concentration 'of the fluids of the body tissues are definitely less soluble in those fluids than is ordinary Insulin. Such a product, which we call an Insulin-protamine composition, may safely be injected either subcutaneously or. intramuscularly, (desirably not intravenously if the slow effect is desired, for on intravenous injection the efiect of our product is substantially the same as that of ordinary Insulin) and if at or near the hydrogen ion concentration of e the fluids of the bodytissues is not immediately entirely taken up from the site of injection by such fluids; but instead, in slowly decreasing amount, remains at the site of injection for a considerablenumber of hours, during which the Insulin is slowly taken up by the fluids of the body tissues and utilized for a fairly uniform sugar control over a fairly long period.

Protamines, which are relatively simple alkaline proteins ordinarily obtained from the sperm of fish, have been known for a great many years-at least one as early as 1868, when Mie- 5 scher produced salmine. They have been known as protein precipitants since about 1890, when that property was first observed by Kossel, who with his co-workers produced a number of protamines and determined the general nature of their chemical constitution. v

By the term protamine, we mean those very simple basic proteins and protein split-products, commonly obtainable from the sperm and/or testicles of fishes, which in their composition include arginine and may include either or both-histidine and lysine.

Among the protamines which have been combined with Insulin, to produce Insulinprotamine compositions that give the desired re-- suits in varying degree, arethe following:

Clupeine (from herring sperm) Salmine (from salmon sperm) Scombrine (from mackerel sperm) Cyclopterine (from lump-sucker sperm) Sturine (from sturgeon sperm) Salmiridine (from the sperm of Salmo irideus Gibbons, or rainbow trout) Protamines derived from the sperm of the fol-' lowing species of fish:

Scientific name Common name gm ,foniinclir Mitchill Brook trout. imo truita Linn, Forma Fario....-.

River trout.

Coreaonu: animus L1nne European white fish. Belem 0cm Risso Needle fish.

Aim slow Cuvier Allice shad.

Coma mnphu Linn- Sculpin.

Insulin'-scombrine composition, and Insul n salmine composition, made by combining (perhaps chemically combining) Insulin with clupeine, scombrine, and salmine respectively, also have low solubilities in such fluids.

Insu1in-protamine compositions have been used on diabetics of all ages and classes and with varying degrees-of diabetic gravity. Most of the cases have been severe ones, some with various complications. Up to the present time, more than 15,000 blood-sugar determinations and more than 3,000 determinations of sugar and ammonia in the urine have been made in the course of our investigation of Insulin-protamine compositions.

The same patients have at times been treated alternately with ordinary Insulin and with In- 6 sulin-protamine composition; so that the controis were on the same patients who received the Insulin -protamine composition. O'ur investigations show the following: The sharp peak eiIect usually seen three or four hours after the injection of ordinary Insulin is largely and often wholly avoided by the use of Insulinprotamine composition. The effect of Insulinprotamine composition is more prolonged than that of ordinary Insulin; so that necessity for Insulin administration during the night, as is essential in certain cases when ordinary Insulin is used, is eliminated even in those cases. Without changing the number of injections, therefore, we can by using an Insulin'-protamine composi-- tion diminish 'the blood-sugar fluctuations, reduce or suppress th glycosuria and reduce the ammonia excretion, and at the same time reduce the risk of the occurrence of either hypoglycemia or hyperglycemia and we can do this, with effective sugar control throughout the entire twenty-four hours, with a smaller total quantity of Insulin per day than when ordinary Insulin is used.

To date no ill effects from Insulin-protamine composition have been'observed. The injections have been substantially painless, and neither untoward local reactions nor'untoward, protein reactions have been observed. The Insulin action is slower, and is not so high and lasts over a longer period, with Insulin-protamine composition than with Insulin. For this reason, in certain acute diabeticconditions (such as coma) ordinary Insulin is to be preferred if used subcutaneously or intramuscularly, because it works more rapidly. If desired, Insulin-protamine composition and ordinary Insulin may both be administered to the same patient, as by administering the ordi- 'nary Insulin during the day and Insulin-protamine composition at night, or under some conditions by administering both at the same time, either in the same injection or at different locatlons. The dose of Insulin-protamine composition varies with the condition of the patient, and must depend on the discretion of the physician, but ordinarily is based on the amount of Insulin the Insulin-protamine composition contains.

We have found that ordinarily the subjective effect on the patient is better when Insulin-protamine composition is used in the treatment than when only ordinary Insulin is used; because by using Insulin-protamine composition, either solely or in part, the patient may be relieved completely of feelings of uneasiness from acidosis or high blood sugar without requiring injections more frequently than two or at most three per day.

Thus by the use of Insulin-protamine composition the blood sugar may be controlled effectively through the entire twenty-four hours; and

the dangerous diabetic condition of excessive blood sugar between midnight and morning, encountered in most diabetic patients under treatment with ordinary Insulin and making it necessary in many of those cases to administer Insulin during the night, may be completely eliminated.

The preparation of "Insulin-protamine composition is very simple. Ordinary Insulin (usually in the form of Insulin hydrochloride) and the protamine to be used are. simply brought together at a suitable hydrogen ion concentration so that they have an opportunity to interact on each other to produce the desired Insulinpr'otamine composition. Neither the "Insulin nor the protamine is necessarily pure, as -either or both may be commercial or technical products; 5

but purity is desirable, as it promotes stability? interaction between the two components; audit 'i may be that the resultant product involves some kind of physical or chemical combination.

The two components may be in almost any proportions. Indeed, the presence of an excess of either component is in certain instances desirable. Thus an excess of Insulin in the composition gives both a prompt action and a continued action, which is desirable in some conditions; while an excess of protamine in the composition serves as a protein-precipitant 'to precipitate proteins in those fluids of the body tissues which come into contact with the injected material, and thus to reduce the solvent-power of those fluids for the Insulin'-protamine composition.

Usually the two components are brought together in solution, conveniently but not necessarily in water solution, as interaction is thereby facilitated; and it is desirable that the solution as administered be bufiered, with a non-toxic buffer, to a hydrogen ion concentration which is approximately equal to that of the fluids of the body tissues, or between pH 6.7 and 8.0. The buffering need not be and desirably is not until just prior to administration. The bufiering can be obtained, for example, by appropriate concentration of the proper phosphates or borates, in known manner.

A highly satisfactory way of bringing about the reaction is to dissolve the two components in separate quantities of water, the Insulin in slightly acid water and the protamine in slightly alkaline water, and then to mix the two solutions, predetermining the hydrogen ion concentrations of the two component solutions so that when they are mixed the final hydrogen ion concentration is in the neighborhood of that of the fluids of the body tissues.

The Insulin'-protamine composition which is thus formed is very nearly insoluble in water at and near the hydrogen ion concentration of the fluids of the body tissues, so that the"Insulin is taken up by such fluids at a very slow rate when and after it is injected. Because of this high degree of insolubility, it is desirable that the material prepared as above outlined should be well shaken immediately prior to injection, to be sure that all the Insulin-protamine composition is in suspension and will be administered. We have noted no ill efiects from the injection, in

spite of the fact that it is largely of a suspension.

The following examples show methods of making Insulin-protamine composition:

Example A.10 grams of ordinary Ins in and 1 to 5 grams of salmiridine are dissolve in 6 liters of water-the Insulin in 5 liters and .the salmiridine in 1 liter-and the two watery solutions are simply mixed. The Insulin solution is desirably adjusted to about pH 2.75; and the saliniridine solution is adjusted to about pH 8.0, .as by the addition of an appropriate phosphate buffer. When the two solutions are mixed this produces a hydrogen ion concentration of about pH 7.4. The resultant product, Insulin-salmiridine composition, is a suspension, is administered as such, and its dosage is that amount which contains the quantity of Insulin per se that it is desired to administer.

Example B.10 grams of ordinary Insulin and 1 to 5 grams of clupeine are dissolved in 6 liters of water-the Insulin in 5 liters and the clupeine in 1 liter-and the two watery solutions are mixed. The hydrogen ion concentrations are suitably adjusted, as in Example A. The re--- sultant product, Insulin-clupeine composition, is a suspension, is administered as such, and again the dosage is that quantity which contains the amount of Insulin per se that it is desired to administer.

In both of these exampleaand others using difierent protamines (including those listed above) in the same general way, the concentrations of thesolutions may be varied over a very wide range. Thus instead of dissolving the 10 grams of "Insulin in 5 liters of water, as suggested, that quantity of Insulin may be dissolved in any desired quantity of water, depending upon the ultimate concentration desired. Similarly, the amount of water used to dissolve the protamine may be varied as desired.

We are not yet fully informed as to the stability of Insulin-protamine compositions over long' product in one ampoule; and in a companion ampoule to put a buffer solution of such quan-' tity and alkalinity that when it (or a proper amount of it) is mixed with the contents of the first ampoule a hydrogen ion concentration of about pH 6.? to 8.0 is produced. The buffer solution may be .a suitable phosphate solution. Be-

fore administration, appropriate amounts of the contents of the two ampoules are suitably mixed, and the mixture injected into the patient.

A third way is to put a mixture of the Insulin and the buffer in one ampoule,- and to put a solution of the protamine in another ampoule, with the contents of both ampoules at about pH 7.4. The contents of the ampoules are appropriately mixed just prior to administration, and the mixture (which is a suspension) is injected into the patient.

Still another way is to have ampoules in sets of three, one ampoule with the ordinary Insulin, the second with the protamine, and the third with the buffer solution. These also will be appropriately mixed'prior to administration, and the mixture injected as before. This, however, in-

volves more complexity than is usually desired or necessary.

Although we prefer that the protamine and the Insulin shall be kept separate until shortly before administration, yet it is entirely feasible, or at least certainly feasible if the material is not to be kept too long before use, to put the product up in single ampoules, containing the Insulin-protamine composition in a buffered solution. Such buffered solutions of Insulinprotamine composition have been kept for weeks without apparent loss of potency.

Eiether the protamine or the Insulin, or both of them, may be in solid form at mixing, and the liquid necessary for injection may be provided in any other way, either during the mixing or at any time prior to administration.

Instead of using water. as the solvent, for one or more of the ingredients, we may use other solvents; such for instance as alcohol, ethylene glycol, propylene glycol, and glycerine, of varying concentrations, or indeed any non-toxic solvent in which the Insulin-protamine composition is substantially a suspension rather than a solution. We claim as our invention: 1. The resultant product obtained by mixin Insulin and a protamine.

2. :I'he product obtained by bringing Insulin and a protamine together, in a bufl'ered solvent.

having a hydrogen ion concentration in the vicinity of that of the fluids of the body tissues.

3. An Insulin'-protamine composition.

4. An Insu1in-salmiridine composition. 5. An Insulin-clupeine composition. 6. An Insulin-sa1mine composition. 7. "The process of making an Insulin product which at the hydrogen ion concentration of the HANS CHRISTIAN HAGEDORN. BIRGER NORMAN JENSEN. INGRID WODSTRUP-NIELSEN. 

